Cardiac glycoside-elicited inotropy is known to be due to inhibition of the Na,K-ATPase. This laboratory discovered the existence of Na,K-ATPase isoforms in the brain, and that they had markedly different affinities for cardiac glycosides in some species. In the first period of support, monoclonal antibodies were produced that permitted identification of three Na,K-ATPase isoforms in the heart. The monoclonal antibody epitopes were mapped; used to determine specificity; and used to study transmembrane protein topography. They then were used to assess factors that control the expression of the isoforms in the rat heart. Changes were seen during development, hypothyroidism, and hypertension that are important for understanding the heart's physiological sensitivity to the drug. The objective now is to use monoclonal antibody epitope mapping to investigate the structure of the Na,K-ATPase isoforms. Antibodies will aid the biochemical analysis of isoform variants. They will be used to determine the topography of transmembrane segments in the controversial C-terminal half. They will also be used to deduce the location of the ouabain binding site. Such physical evidence complements and tests models supported by molecular biology; for example, we have evidence that the ouabain binding site is not located at the position indicated by site-directed mutagenesis.